This proposal describes studies of enzymatic catalysis using the methodology of Raman difference spectroscopy. Specific interactions between an enzyme and its substrate are responsible for both the enormous catalytic power of enzymes as well as the fact that enzymes conduct the correct chemical reaction. How small molecules bind to proteins is of central importance to Biology and Medicine, in general, and understanding binding in molecular terms is key to understanding enzymic mechanism, specifically, and to the drug discovery process as well. Raman difference spectroscopy, as developed in our work, permits measurement of the vibrational spectrum of specific molecular groups within proteins, and this yields a high resolution, often unique, characterization of the electronic structure in bonds and between atoms which is very useful in understanding the noncovalent interactions that take place between an enzyme and its substrate. The general approach taken here is to characterize, one by one, the internal coordinates that make up the reaction coordinate, relating results to catalytic rates in order to understand mechanism. Two enzyme classes will be studied: the NAD(P)-linked enzymes and those that are involved with phosphoryl transfer reactions. Within the first class, lactate dehydrogenase and dihydrofolate dehydrogenase will be studied in depth. Our studies to date have characterized the keto side of the lactate dehydrogenase, and the alcohol side of the reaction is now to be extensively studied. The pKa dependence of hydride transfer in dihydrofolate reductase will be probed by measurements of the active site carboxyl group. The C4 atom of bound NAD(P) cofactors either donates or yields a hydride ion to substrate in the NAD(P)-linked enzymes. The frequency of the key C4-H coordinate of the cofactor will be measured in selected enzymes and in model compound systems to understand how enzymic bond distortion is-accomplished and regulated across this large class. Studies of phosphoglucomutase will be continued by measurements of phosphate substrates and vanadate transition state analog bound to mutant forms of the protein involving changes to the binding site ionic residues. This will probe the mechanism of phosphate binding to the protein, and this may be useful to understanding binding of phosphate to other phosphate binding proteins. In addition, the data will be used to appraise how the active site is arranged to provide the enormous catalytic power of this enzyme, whose mechanism is very different from model solution chemistry. In parallel studies, substrate and transition state analog binding to Rnase will also be investigated because of the similarities and differences to the phosphoglucomutase system. The ATPase mechanism of the p2l protein, mutants of which are associated with a large number of human tumors, will also be studied. Several studies of model phosphate systems will be studied in order to understand our results and the results of 18O kinetic isotope effects.